Download Analysis of GDSL lipase (GLIP) family genes in rice (Oryza sativa)

POJ 5(4):351-358 (2012)
ISSN:1836-3644
Analysis of GDSL lipase (GLIP) family genes in rice (Oryza sativa)
Yunyun Jiang, Rongjun Chen *, Jiali Dong, Zhengjun Xu, Xiaoling Gao
Rice Research Institute of Sichuan Agricultural University, Chengdu, Sichuan 611130, China
*Corresponding author: [email protected]
Abstract
Lipase is one of the lipid hydrolyzing enzymes, distributed broadly throughout plants, animal and microorganisms. GDSL-lipases is
one of the lipases that exhibit a GDSL motif GxSxxxxG, in which the active site Serine is located near the N-terminus and display a
Gly-Asp-Ser-(Leu) [GDS(L)] motif in conserved block I. However, the knowledge about their roles in developmental processes and
response to various stimuli are still very limited in rice. A systematic analysis revealed the presence of at least 113 GDSL lipase
(GLIP) genes in the rice genome. The tandem gene duplications have contributed a major role in expansion of this gene family.
Phylogenetic analysis classed proteins into three groups; OsGLIP group B contained 56 genes, 50 in group A and only 2 genes in
group C. The organization of putative motifs indicated potential diverse functions of GLIP gene family members in rice. Microarray
data analysis revealed tissue and developmental stage-specific expression patterns of several OsGLIP genes. 38 OsGLIP genes were
especially expressed in stigma and seed germination, several genes expressed exclusively in root and 17 OsGLIP genes were induced
by any of three stresses. Our analysis also suggests differential accumulation of cluster genes during these processes. Our analyses
indicated OsGLIP genes may have potential roles in rice development and abiotic stresses.
Keywords: GLIP, Oryza sativa, chromosomal localization, phylogenetic analysis, expression patterns, microarray.
Abbreviations: MeJA - methyl jasmonic acid SA -salicylic acid ABA-abscisic acid
Introduction
Lipase is one of the lipid hydrolyzing enzymes, distributed
broadly throughout plants, animals, and microorganisms. Three
highly conserved amino acid residues (Ser, Asp, and His),
which form the catalytic triad, are present in lipase family
members (Arpigny and Jaeger, 1999; Hong et al., 2008). A
pentapeptide consensus motif (Gly-X-Ser-X-Gly) is found in
members of the lipolytic enzyme family, which includes lipases
and esterases. Lipases preferentially hydrolyze long-chain
acylglycerols (10 carbon atoms at least), whereas esterases are
specific for short-chain acylglycerols (10 carbon atoms at most)
(Ling et al., 2006). GDSL lipases represent a subfamily of
lipolytic enzymes, which like other members of the lipase and
esterase families, possess a conserved catalytic triad (Akoh et
al., 2004; Lee and Cho, 2003). Most lipases contain a GxSxG
motif, but GDSL lipases exhibit a GDSL motif GxSxxxxG, in
which the active site Ser is located near the N-terminus and
displays a Gly-Asp-Ser-(Leu) [GDS(L)] motif in conserved
block I (Brick et al., 1995; Lee et al., 2009; Updegraff et al.,
2009; Upton and Buckley, 1995).
All of GDSL enzymes have a consensus sequence that is
divided into five conserved sequence blocks (I–V). The GDSL
family is further classified based on the strict conservation of
the catalytic residues S, G, N, and H within conserved blocks I,
II, III, and V, respectively (Dalrymple et al., 1997; Ling et al.,
2006; Mølgaard et al., 2000; Lo et al., 2003). GDSL-lipases are
involved principally in the regulation of plant development,
morphogenesis, Enod8 is a member of the GDSL
lipase/esterase family that is isolated from Medicago sativa
root nodules (Pringle and Dickstein, 2004). BnLIP2 (Brassica
napus.L) encodes a GDSL lipase which is induced during
germination and maintained in mature seedlings (Ling et al.,
2006). GDSL lipases also perform critical roles in the biotic
and abiotic stress responses of plants. Br-sil1 (Brassica rapa
salicylate-induced lipase-like 1) is induced by salicylic acid and
pathogens (Lee and Cho, 2003). GLIP1 is an Arabidopsis
GDSL lipase that possesses anti-microbial activity and
regulates pathogen resistance to Alternaria brassicicola in
association with ethylene signaling (Hong et al., 2008; Oh et al.,
2005), GLIP2 playes a role in resistance to Erwinia carotovora
via negative regulation of auxin signaling (Lee et al., 2009; Yu
et al., 2010). In hot pepper, CaGLIP1 confers resistance to
Xanthomonas campestris pv. vesicatoria that has been shown to
modulate pathogen and wound stress resistance, and is induced
in by MeJA (Hong et al., 2008; Kim et al., 2008; Park et al.,
2001).
Over-expression of AtLTL1 is induced by SA treatment
increased salt tolerance and other environmental stresses in
yeast and Arabidopsis (Hong et al., 2008; Naranjo et al., 2006;
Riemann et al., 2007). In addition, GDSL-lipases are indicated
as participating in hormonal pathways related to growth
processes (Kiba et al., 2005; Naranjo et al., 2006). The
researches of GER1 (rice), RGE1 (Arabidopsis) and EXL4
(Arabidopsis) also evidenced a correlation between the
expression of GDSL-lipases and other developmental processes
(Kondou et al., 2008; Riemann et al., 2007; Updegraff et al.,
2009). Partial GDSL-lipases involve in the metabolism of cutin
and wax (Broun et al., 2004; Kim et al., 2008; Kondou et al.,
2008; Panikashvili et al., 2010; Riemann et al., 2007; Takahashi
et al., 2010). An unrooted phylogenetic tree is constructed from
the amino acid sequences of 604 GDSL lipases from seven
species (Arabidopsis thaliana, Populus trichocarpa, Vitis
vinifera, Orysa sativa, Sorghum bicolor, Picea sitchensis,
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Table 1. Putative motifs predicted in OsGILP proteins.
Group A
Group B
Block I
FGDSxVDxGNNN
FxFGDSxxDTGN
Block II
TGRFSNGxxxxD
PTGRxSDGRLxxDF
Block III
SLFxxxxGxxND
SLFxxGEIGGNDY
Block IV
GARxxxxxxxxPxGCxP
GARxxVVPGxxPxGCxP
Block V
FWDxxHPTEAAN
SWDGxHxTEAAY
Significant motifs (e-value <e-100) of more than 10 amino acid length present in at least 20 GILP proteins
were predicted by MEME research. The consensus sequence GILP proteins containing are given.
Fig 1. Chromosomal distribution of OsGILP genes. Graphical represent of the location of different GILP genes IDs on rice
chromosomes. The genes labeled on left side are oriented from bottom to top and those on right side are oriented from top to bottom
on rice chromosomes. The genes localized on duplicated chromosomal segments are connected by blue and red dashed lines. Fifteen
clusters (three on chromosome 1, 2, 5 and 6, one on chromosome 3, 7 and 10) of tandemly arranged OsGILP genes are indicated in
gray rectangles and duplicated-pairs in boxes. The chromosome number is indicated at the bottom of each chromosome.
Physcomitrella patens). The topology of the tree depicts two
major and one minor subfamily. This division is also supported
by the unique gene structure of each subfamily (Volokita et al.,
2011). However the roles of OsGLIP genes in development and
abiotic stresses are rarely reported.
third cluster of 2 members of group A. Chromosome 3, 7 and
10 have one cluster of GILP genes, all members were
belonging to group B. This data suggested the major
contribution of tandem duplications (60 of 113, 53.1%) to the
GILP family expansion. On the other hand, we found 13
members of GILP family located on the segment-duplicated
regions. Since the number of GILP genes located on
segment-duplicated regions was less than those present in
tandem, tandem duplications appear to have a major role in
expansion of this gene family.
Results and Discussion
Identification of genes encoding GILP proteins in rice and
tandem duplications
From TAIR and GRAMNENE database, one hundred and
thirteen GILP genes were obtained (latest data). The
comparison with Volokita et al. (2011) research shows that 108
genes were identical, while others had discrepancy. To
investigate the contribution of gene duplication in the
expansion of GILP gene family, the chromosomal location of
each GILP gene was determined based on the information
provided by RGAP. These GILP genes were located in all 12
chromosomes (Fig. 1). The position of gene impacts the
function, most GLIP genes were in cluster at arms of
chromosomes, a few at the telomeric ends and near centromere.
The rice genomes have undergone several rounds of
genome-wide duplication events, including polyploidy, which
have great impact on the expansion of a gene family in the
genome. The chromosomal localization of most members of
this family indicated a non-random distribution. As these genes
were located within ≤5 genes in between them, possibility for
tandem duplication was analyzed using Vista Tools for
Comparative Genomics (Frazer et al., 2004). A total of fifteen
clusters of tandem arranged genes were observed on different
chromosomes. Chromosome 1 and 5 harbored three clusters of
GILP genes (29 members of group B), respectively.
Chromosome 2 contained three clusters of 7 GILP genes (all
members of group A). Chromosome 6 has three clusters of 8
GILP genes, first and second cluster of 6 members of group B,
OsGLIP genes phylogenetic analyses
All of 113 GDSL-lipases homologous proteins were retrieved
for phylogenetic analyses using their full-length protein
sequences. Based on the protein sequence alignments and
evolutionary relationships, the largest number of OsGLIP genes
(56) were included in group B followed by 50 genes in group A,
and one minor group C with just 2 members. This result was in
agreement with Volokita et al. (2011) (Fig. 2). The additional
putative conserved motifs in GLIP proteins were investigated
using MEME program. The results show that there were five
consensus sequences in Block I-V (Table 1). The GDSL motif
was found to be close to the N-terminus. The sequences
FGDSxxDxG are more conservative in rice, Ser in Block I, Asp
in Block III or Asp and His Block V, putatively constituting the
catalytic triad SDH. The exact location of active residue Asp
has not been exactly confirmed yet (Ling et al., 2006; Upton
and Buckley, 1995; Volokita et al., 2011). Most notably the
numbers of Block V was larger than Block III in rice. There
were no specific to the members of a particular class, the same
blocks contained different conservative ammo acid residues
within groups (Table 1). These putative conserved sequences in
same motif might provide diversity in functions of the OsGLIP
proteins.
352
Table 2. Putative regulatory cis-element sequences in 5'-upstream regions of 11 OsGILP genes.
Name
Description
LTR
cis-acting element involved in low-temperature responsiveness
CCAAT-box
MYBHv1 binding site
CGTCA-motif
cis-acting regulatory element involved in the MeJA-responsiveness
TC-rich repeats
cis-acting element involved in defense and stress responsiveness
ABRE
cis-acting element involved in the abscisic acid responsiveness
ARE
cis-acting regulatory element essential for the anaerobic induction
O2-site
cis-acting regulatory element involved in zein metabolism regulation
TCA-element
cis-acting element involved in salicylic acid responsiveness
TGA-element
auxin-responsive element
TGACG-motif
cis-acting regulatory element involved in the MeJA-responsiveness
motif IIb
abscisic acid responsive element
ERE
ethylene-responsive element
HSE
cis-acting element involved in heat stress responsiveness
MBS
MYB Binding Site/MYB binding site involved in drought-inducibility
CE1
cis-acting element associated to ABRE, involved in ABA responsiveness
P-box
gibberellin-responsive element
GARE-motif
gibberellin-responsive element
TATC-box
cis-acting element involved in gibberellin-responsiveness
TATCCAT/C-motif
cis-acting regulatory element; associated with G-box like motif; involved in sugar
repression responsiveness
GC-motif
enhancer-like element involved in anoxic specific inducibility
motif IIb
abscisic acid responsive element
as-2-box
involved in shoot-specific expression and light responsiveness
GCN4_motif
cis-regulatory element involved in endosperm expression
Skn-1_motif
cis-acting regulatory element required for endosperm expression
Fig 2. Phylogenetic OsGILP proteins and their classification. The unrooted tree was constructed based on multiple sequence
alignment of full-length protein sequences using ClustalX program by Neighbor-joining method with 1000 bootstrap replicates. All
the OsGILP proteins are classed into three groups. The bar indicates 0.05 substitutions per site.
7-day old shooting stage (cold, salt and desiccation) (Fig. 4)
and different reproductive development under three stresses
(cold, high temperature and desiccation) (Fig. 5).
Lipases/esterases were active in hydrolysis and synthesis of
abundant ester compounds. Extracellular lipases (EXL1-6) were
isolated from A. thaliana pollen coat, anther-specific
proline-rich protein genes (APGs) were from A. thaliana and B.
napus, BnLIP2 (B. napus L.) was a tissue-specific expressing
gene during reproductive growth and strongly expressed during
seed germination (Clauß et al., 2008; Clauß et al., 2011; Hong
et al., 2008). 38 OsGLIP genes were highly expressed in stigma
indicating that these genes might be expressed in stigma
specifically. The expression of many OsGLIP genes was
restricted to a particular tissue or developmental stage, whereas
Expression of GLIP genes during development and under
abiotic stress conditions in rice
Although the roles of AtGLIP genes have been explored in
various stress responses, the evidences for their role in rice
growth and development were very limited (Hong et al., 2008;
Kim et al., 2008; Kondou et al., 2008; Lee et al., 2009;
Panikashvili et al., 2010; Updegraff et al., 2009; Volokita et al.,
2011). The study of gene expression patterns of all the
members of a gene family provided insight into their functional
diversification. The clustering of expression profiles of 112
OsGLIP encoding genes were collected from a microarray
based transcriptome profiling of 23 stages of vegetative and
reproductive development (Fig. 3), three stress conditions at
353
a few OsGLIP genes exhibited expression in wide
developmental stages. For example, the expression of one gene
(LOC_Os02g01980) was restricted to panicle development
stages (P3-P6). Eleven genes were expressed preferentially in
shoot apical meristem tissue and panicle development stages
(P1-P5), 27 genes expressed exclusively in root (Fig. 3). These
genes may perform specific roles in these tissues or
developmental stages. To reveal the functional redundancy or
diversification of duplicated OsGLIP genes, their expression
patterns were analyzed. The expression patterns of duplicated
genes indicated their evolutionary fates. Among the fifteen
gene pairs localized on duplicated chromosomal segments, four
pairs of genes LOC_Os01g46080/090, LOC_Os01g46120/169,
LOC_Os05g11970/950 and LOC_Os05g43100/120 exhibit
similar expression patterns, whereas other pairs showed
divergent expression patterns. The results suggest the evolution
of GLIP gene family have occurred by gene duplication
followed by retention due to sub- or neo-functionalization of
the duplicated genes. Many GDSL-lipases genes have been
implicated in various abiotic stress responses in plant species
(Clauß et al., 2008; Clauß et al., 2011; Lee et al., 2009;
Takahashi et al., 2010).
Our analysis showed that 42 members were with high signal
value (control group signal value > 100) for rice seedlings
treated with different abiotic stresses (desiccation, salt and cold)
as compared to mock-treated control (7-day-old seedlings)
(Fig. 4), 8 were found to be up-regulated by more than 2-fold
and 15 down-regulated at desiccation stress (Fig. 6 A, B). In
7-day-old rice shooting, genes were more sensitive to
desiccation stress, 14 down- and 7 up-regulated significantly.
Meantime, 7 genes were down-regulated and 2 induced in salt
test. However, just 3 genes were down-regulated and none
up-regulated under cold condition (Fig. 6A, B). In Fig. 5, a
total of 43 genes with high signal value (control group signal
value > 100), 11 were found to be up-regulated and 10
down-regulated at all stresses by more than 2-fold (Fig. 6C, D).
Six genes were specifically down-regulated in leaves in cold
stress at booting stage and high temperature at
heading/flowering stage, in which 4 of OsGILP were observed
to be down-regulated in shooting leaves during cold stress; 3 in
booting leaves and spike in high temperature stress; 2 in
booting leaves at high temperature and desiccation and in
heading/flowering spikes at cold; and none in
heading/flowering spikes at desiccation and high temperature
stresses. All of these genes showed high level expression in
stigma and low at SAM, P2 and S3-S5 stages (Fig. 4).
Microarray analysis performed on seedling, booting, heading
and flowering stages subjected to cold, dehydration and high
temperature stress revealed eleven 2-fold up-regulated genes in
at least one of the stress conditions. In seedling leaf only one
gene (LOC_Os01g11570) was induced expression by cold that
was rather expressed at high level, on the other hand two genes
(LOC_Os01g11650 and LOC_Os06g34120) were expressed at
low level suppressed in young leaf. At booting stage two genes
under dehydration stress, 6 genes were induced.
At heading and flowering stage, 4 genes were induced by
high temperature in spike. The expression pattern of cluster
genes was not completely identified in response to abiotic
stresses. For example, the fist cluster (LOC_Os01g11710/730)
genes in Fig. 6A were down-regulated under desiccation stress,
while only LOC_Os01g11730 was changed under salt and cold
condition more than 2 fold. The second gene cluster
(LOC_Os01g46090/120/169) was also down-regulated under
desiccation stress, but only one gene (LOC_Os01g46120)
suppressed by salt treatment. All 3 clusters showed no change
under cold stress. In the third cluster genes
(LOC_Os06g34070/120) the expression responses to
Fig 3. Differential expressions of GLIP gene family members
in various tissues and developmental stages. Hierarchical
clustering analysis of 112 OsGILP genes represented on
Affymetrix Rice Genome Array is shown. For clustering we
used average log2 signal values for three biological replicates of
each sample after normalization of the raw data. The color
scale for log signal values is shown at the bottom. SAM, shoot
apical meristem; P1-P6, stages of panicle development; S1-S5,
stages of seed development. The series GSE6893 includes
microarray data from 45 hybridizations representing three
biological replicates each of 15 different tissues/organs and
developmental stages, three biological replicates were available
only for stigma and ovary in the series GSE7951 dataset.
354
desiccation stress was observed while no change under salt
condition detected. In this cluster only one gene were
down-regulated by cold stress. Two cluster genes induced by
any stress in Fig. 6B. The first pair of genes
(LOC_Os05g11910/950) both induced by desiccation but not
under cold stress. The second cluster genes, one
(LOC_Os06g50940) induced by desiccation, another
(LOC_Os06g50950) by salt stress. No gene of cluster was
down-regulated under different reproductive development
stages under cold, high temperature and desiccation stresses
(Fig. 6C). Fig. 6D shows that one cluster genes
(LOC_Os01g46080/090/169) were significantly induced by
stresses, in which all of 3 genes were up-regulated in booting
spikes under desiccation stress. Only LOC_Os01g46080 was
induced in booting leaf at desiccation, while LOC_Os01g46169
was suppressed in leaf at high temperature and
LOC_Os01g46090 in spike at heading/flowering stage under
high temperature circumstances. The analysis suggested that
expression of different members of the OsGILP gene cluster
was regulated differentially after abiotic stresses in different
tissues, which might be attributed to their tolerance or
susceptibility behavior to stress.
Analysis of cis-elements in 17 abiotic stress responding genes
Fig 4. Differential expressions of OsGILP genes in response to
three abiotic stresses. Hierarchical clustering of 42 OsGILP
genes showing the significant differential expression under
three abiotic stress conditions. DS, desiccation stress; SS, salt
stress; CS, cold stress; CK, control (7-days old seedlings, signal
value > 100). The color bar representing log2 signal values is
shown at the bottom.
To investigate the probable explanation for the 17 abiotic stress
up-regulated genes, we analyzed their promoter sequences,
about 1.5 kb upstream from translational start site. The results
of Plant-CARE database and the PLACE databases shows that
a total of 21 types of CREs were identified responding to
abiotic stress, which contained 3 kinds of growth regulatory
elements (as-2-box, Skn-1_motif and GCN4_motif) (Table 2).
The names and functions of these 24 motifs were shown in
Table 2. Their locations on upstream sequences are displayed
in Fig. 7.
TGACG-motif, ABRE and ARE motifs were found in 12, 11
and 10 out of 17 genes, respectively. In reverse, the motifs CE1,
motif IIb and TATC-box were just found in one gene. Other
motifs distributed in 3 to 9 genes, apart from GC-motif
stretching in 2 genes. Totally 9 genes contained 6-8 CREs, one
gene (LOC_Os01g42730) had the biggest number of 14,
following 2 genes (LOC_Os01g42730 and LOC_Os06g50940).
There were many phytohormone auxin responsive cis-acting
elements, such as MeJA, ABA, zein, ethylene, gibberellins.
This analysis revealed that the regulatory elements were
more conserved in duplicated OsGILP genes with similar
expression patterns (for example, LOC_Os05g11910/950 and
LOC_Os06g50940/950) as compared to the GLIP genes with
divergent
expression
patterns
(such
as,
LOC_Os01g46080/090/169). The difference in the regulatory
elements of duplicated genes might explain their divergent
expression patterns. However, the existence of some other
regulatory mechanism, which is responsible for divergent
expression patterns and/or non-functionalization of one of the
duplicates, could not be ruled out.
Fig 5. Differential expressions of OsGILP genes in response to
various biotic stress conditions. Hierarchical clustering of 43
GST genes showing significant differential expression in
differential developmental stages under various abiotic
conditions is shown. 1, seedling stage; 2, booting stage; 3,
heading and flowering stage; L, leaf; S, spike; CK, as compared
with an untreated rice (signal value > 100 in any tissue); CS,
cold stress; DS, dehydration stress; HS, high temperature stress.
The color bar representing log2 signal values is shown at the
bottom.
Materials and methods
Plant materials and growth conditions
The viable seeds of cultivated rice Pei’ai 64S (Oryza sativa L.
indica) were suspended in a sterile solution of 0.1% HgCl2 for
10 min, washed 3 times using running water, immersed for 3
days under 25 oC and water changed once a day, then were
germinated and grown in distilled water at 37 oC for 2~3 days.
They were sown in batches in pots that put in net basin at
Institute of Subtropical Agriculture of Chinese Academy of
355
Fig 6. Genes up-regulated or down-regulated in any abiotic stresses. The genes are down- or up-regulated by more than 2-fold in
response to various biotic stress conditions is compared with CK have been shown. The color rectangles represents gene. Y axis is
changed fold; X axis is different tissues under various stresses. Dashed line represents expression down- or up-regulated 2 fold.
Science. Plants were divided into one control and three
treatment groups. The control group was maintained under
natural conditions, regular water and fertilizer management,
pest and disease control. The treatment groups were exposed to
drought, heat and cold stresses. At five-leaf stage, part of them
was taken as the test material of the seedling stage. Other parts,
as test material of booting and flowering stage are transplanted
to the other pots, 5 plants from each pot.
sealed the tubes with Parafilm wrap. The samples were stored
at -70 oC until required.
Total RNA isolation
Total RNA was isolated from the frozen samples using TRIzol
(Invitrogen). The samples saved in -70 °C were taken out,
vortexed to homogeneity, chloroform (200 μL) was added,
vigorously shaken for 15 s, and then centrifuged at 12 000 × g
for 15 min at 4 °C. The upper layer was carefully removed
from each tube, was transferred to another centrifuge tube.
Then the isopropanol (500 μL) was added, precipitated for at
least 1 h at -40 °C, then centrifuged to separate the RNA. The
RNA pellets were twice washed by 75% ethanol, air dried and
dissolved in the appropriate volume of RNase-free water. The
purity of RNA was determined by the A260/280 absorbance
ratio (1.9-2.0). Isolated RNAs were stored at -70 °C, after
checking the purity and integrity of 18 S, 5 S and 28 S rRNA
bands on 1.5% agarose gel.
Rice cold, heat and water-deficit treatment
For the drought tests the watering was avoided to the basin
until the treatment group dried out. Meanwhile, the control
group was also put in the dry shed, but with water in pots, then
the leaves were harvested when they started curling after 16 h.
For the heat tests, materials were put in climate incubator,
PGC15.5 (Percival, USA) for 2 h under 45 oC, while the control
group was put into PGC15.5 under 45 oC. For cold test, we
placed the seedlings into PGC15.5 for 12h under 4 oC at
booting and heading stage for 16h under 12 oC. The control
group was placed into another PGC15.5, and both control and
treatment group was under dark conditions.
Microarray
Affymetrix expression microarray experiments was done
according to manual provided by GeneTech (Biotechnology
Limited Company, Shanghai, China) briefly by the following
steps: (1) Total RNA extraction and purification; (2) cDNA
synthesis and purification; (3) transcription cRNA synthesis
and in vitro cRNA purification (4) cRNA fragmentation,
hybridization solution preparation (5) chip hybridization (6)
elution chip (7) scan chips (8) data analysis.
Raw materials and sample preparation
Four or five countdown second leaves were collected from
treatment and control group, and four or five leaves were
harvested, which were not out of the young panicle or the
middle of spiked out flower. The materials were cut into pieces,
then ground into a dry powder with liquid nitrogen, and
immediately divided into pre-installed 1.0 mL TRIzol
extraction (Invitrogen) in 1.5 mL centrifugal tubes, about 100
mg each tube. We used low temperature marker pen to mark
labels, tightly closed lid, then vigorously vortexed to make sure
that samples were mixed up with TRIzol extraction and then
Sequences analysis
Protein sequences of rice GDSL lipase-like (GLIP) were
acquired from a query search in GRAMENE and rice genomes
356
Expression analysis
We used the Gene Expression Omnibus (GEO, accession
number GPL2025) database for expression data of reproductive
development (GSE6893 and GSE7951) and expression data for
stress treatment (GSE6901) (Jain et al., 2007) of the rice GLIP
gene family. The Affymetrix CEL files of each were imported
and analysed using Gene Chip Robust Multi-Array method.
The IDs of probe sets present on the Affymetrix rice genome
array representing the GLIP genes were identified using the
Rice Multi-platform Microarray Search tool (Saeed et al.,
2003). Cluster analysis on rows (using log transformation) was
performed by, Average Linkage rule of Hierarchical clustering
method.
Conclusion
This study provided not only an updated genomic positions,
phylogenetic relationships and protein structures of the
GDSL-lipase family in rice, but also the identification of
several tissue- and/or developmental stage-specific and abiotic
stress-responsive OsGILP genes included in various classes.
Our results provided a very useful framework and starting point
for revealing the function of GILP family members in rice,
especially those involved in specific developmental processes
and stress tolerance.
Acknowledgments
This research was supported by Fund 11ZA090 (A Project
Supported by Scientific Research Fund of Sichuan Provincial
Education Department) to RJ Chen; we thank Dr. XJ Xia for
help in microarray analysis.
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Fig 7. Distribution of cis-element sequences in 5'-upstream
regions of OsGILP genes. Predicted cis-element sequences are
on 5'-upstream regions of 17 up-regulated OsGILP genes. The
sequence is about 1500bp from the start coden ‘ATG’. Long
line represents DNA sequence; little bar is cis-element.
(TIGR version 6.1) with the key word of GDSL-like
lipase/acylhydrolase. GLIP motif scan was performed using
InterProScan and PFAM Database searches with filter off. We
identified the protein motifs of GLIP genes using MEME
(http://meme.sdsc.edu/meme/meme.html) with the motif length
setting at 6-20, motif for 5. GLIP genes were mapped on
chromosomes by identifying their chromosomal position given
in TIGR rice database. The genome organization and map
location were investigated by the corresponding genome
sequence
with
the
map
viewer
at
NCBI
(http://www.ncbi.nlm.nih.gov/mapview/). The amino acid
sequences of GLIPs were initially aligned using the program
ClustalW (version 1.83) and computing pairwise distance. The
unrooted phylogenetic tree was constructed by the
Neighbor-Joining method and displayed using the same
software MEGA (version 5.02). Bootstrap analysis was
performed using 1000 replicates, p-distance Method,
pairwise-deletion gaps data treatment. DNA sequences of rice
GLIP genes and 1500 bp ahead of the translation initiation
codon (ATG) were collected from the GRAMENE database.
The promoters of GLIPs were analyzed by Plant-CARE
database, and validated the results using the PLACE databases.
357
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